1. Field of the Invention
This invention relates generally to apparatus for the stepwise performance of chemical reactions, and more particularly to apparatus for sequencing proteins and/or peptides.
2. Description of the Related Art
The sequence of amino acids in a protein, i.e. the linear succession of the individual amino acids, is a deciding criterion for its higher structure and, subsequently, for the biological function of the protein. The sequencing of proteins is thus of considerable importance in biochemical research and industry such as, for example, genetic engineering. As used herein no distinction is made between proteins, peptides and polypeptides: the whole group of these substances is addressed when the term "protein" is used.
A protein sequenator for the automatic determination of amino acid sequences in proteins is known from P. Edman and G. gg, European Journal of Biochemistry, Vol. 1, 1967, pages 80-91. This sequenator operates on the principle of the phenylisothiocyanate degradation scheme. According to this degradation process, the free N-terminal alpha-amino group is first converted in a coupling step in a basic environment with phenylisothiocyanate (PITC) into a phenyl thiocarbamyl (PTC) derivative. The phenylthiocarbamyl derivative is cleaved in a strongly acidic environment and is cyclized with formation of an anilothiazolinone derivative (ATZ) which can be converted by treatment with aqueous acid into the more stable phenylthiohydantoin (PTH) derivative. The alpha-amino group of the following amino acid in the chain of amino acids is thus set free and can be subject to a further degradation step similar to the one described. The PTH amino acid can be identified using liquid chromatography.
The sequenator known from the above-mentioned article by Edman and Begg, which is also known from U.S. Pat. No. 3,725,010, comprises a reaction vessel wherein the degradation of proteins to be determined takes place and a plurality of reservoirs for storing reagents and solvents. The reservoirs are under a constant pressure and, by opening appropriate valves, a liquid or gas stream can be fed to the reaction vessel. The reaction vessel is a cylindrical glass cup which spins continuously around its longitudinal axis. The protein is applied as a thin film on the inner wall of the cup. Reagents and solvents entering through a feed line at the bottom of the cup climb up the inner wall due to centrifugal forces and accumulate in a circular groove near the upper rim of the cup. The accumulated liquids are scooped off with a bilge pipe and leave through an effluent line.
In this known sequenator, the application of the protein layer and the guiding of the liquid film of reagents and solvents across the protein layer are not very precise, and comparatively high quantities of protein and liquid are required in order to obtain acceptable reaction yields. Furthermore, as the reagents wherein the proteins are soluble slide across the protein layer towards the circular groove at the rim of the vessel they gradually wash away the proteins.
From U.S. Pat. No. 3,892,531 and from U.S. Pat. No. 4,065,412, devices for sequencing proteins are known which tend to avoid the washing away of the proteins by the solvents and reagents. In these devices, the protein is deposited on a porous surface, for example on a paper strip or on a fibre glass web, and the reagents are applied as vapor. The reaction velocity and the completeness of the reaction, however, are reduced when using the reagents in gaseous or vapor state relative to the liquid state in the Edman process. Furthermore, the protein agglomerates in the cavities of the porous material causing a decrease of the surface of the protein sample and it depends on the properties of the specific protein and on other unforeseen occurrences in which way the protein spreads on the porous surface. As a consequence thereof, there are regions where the protein accumulates so that the accessibility of reagents and solvents is reduced, whereas in other regions there is little or no protein so that the reagents pass there without reaction. These are the main reasons that such gaseous phase methods have comparatively low reaction yields.